Sm2500

The control and the in vivo loaded tibiae from fifteen
mice (n=5 mice/age) were cut in the transverse plane at the midshaft with a
microtome (Leica SM2500S, Munich, Germany) into 2.5 µm thick sections
for FTIRI imaging. The control and the in vivo loaded tibiae
from six mice (n=2 mice/age) were microtomed into 10 µm thick sections
in a direction parallel to the longitudinal axis of the long bones for
synchrotron sSAXS measurements. All sections were imaged with fluorescence
microscopy (Leica DMRB, Munich, Germany), before being measured with FTIRI or
sSAXS.

Tibiae from 12 week-old LC and Gorab^Prx1 mice (n=5/genotype) were dissected, dehydrated in ascending grades of ethanol, embedded in methyl methacrylate, and cut at the mid-sagittal plane with a microtome (Leica SM2500S, Germany) into 2 μm thick sections. The prepared undecalcified tibial sections were mounted on barium fluoride infrared windows for FTIR imaging (Perkin Elmer Spotlight Imaging System, MA). FTIR imaging was performed at the proximal metaphysis and mid-diaphysis of the tibia to remain consistent with the microCT regions of interest. Due to restrictions related to field of view, only the posterior side of these two regions was analyzed. The regions were scanned using a spectral resolution of 4 cm⁻¹ and a spatial resolution of 6.25 μm. FTIR bone parameters calculated were (Boskey and Pleshko Camacho, 2007 (link)): mineral/matrix ratio (area of 916–1180 cm⁻¹/1590–1712 cm⁻¹); carbonate/mineral ratio (area of 852–890 cm⁻¹/916–1180 cm⁻¹); collagen maturity (peak intensity ratio of 1660 cm⁻¹/1690 cm⁻¹); crystallinity (peak intensity ratio of 1020 cm⁻¹/1030 cm⁻¹); acid phosphate content (peak height 1096 cm⁻¹/1128 cm⁻¹).